Escherichia coli phoA gene inactivation, insertion and restoration based on the λ red system

Abstract

The red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in Escherichia coli K-12 through homologous recombination using linear PCR products. Here, E. coli BW 25113 mutants were created by changing the localization of genes on the genome using a method which contains both inactivation and reinsertion steps. The phoA gene, the structural gene for alkaline phosphatase, was inactivated by having the ORF and its regulator region replaced with a kanamycin cassette flanked by FLP recognition target sites. The site for insertion was selected based on the intergenic distance and non-coding regions, thus the new neighboring genes sequences were not affected. The Insertion was carried out by designing primers armed with homologous sequences to the insertion site; the primers contained the enzyme restriction sites in order to ligate the gene PCR product with the FRT-kan-FRT marker prior to the electroporation. The PCR reactions, cultures in the media supplemented with appropriate antibiotics and comparative gene expression study were carried out to confirm the knockout, the insertion and the expression of the gene in its new location. This procedure and the created E. coli mutants can be efficiently used for the study of bacterial genomics, especially in systems biology to understand the relation between the genes loci on the genome and their expression alongside new neighboring genes and others with the same functional group.